The invention relates to an optical arrangement for detecting light emitted by a sample.
The detection of molecules with the help of optical biosensors is done as a rule by detecting a specific binding between the molecules and what are called xe2x80x9creceptor moleculesxe2x80x9d, which are immobilized on a surface. The detection of molecules can be performed very selectively, even from a mixture, by way of such a specific binding. Antibodies or DNA molecules are typical receptor molecules. Typical surfaces are on optical fibers or transparent microscopic slides or cover sheets.
Often, a fluorescent signal specific to the binding between the molecule sought and the receptor molecule is detected in order to detect the molecules. This can be generated by the molecules sought themselves, if they are able to fluoresce.
However, as a rule, what is called a xe2x80x9csandwich testxe2x80x9d is performed, in which the molecule sought is not able to fluoresce, but a third, fluorescence labeled xe2x80x9cprobe moleculexe2x80x9d binds selectively to the binding complex of the molecule sought and the receptor molecule, after this complex has formed.
In order to obtain quantitative information on the quantity of the molecules sought bound to the receptor molecules, only that fluorescence light must be detected that indicates a binding complex between the molecule sought and the receptor molecule. Such a complex is always located close to that surface on which the receptor molecules are immobilized. From this, it follows that the measurement task is to detect selectively fluorescent signals from molecules bound close to the surface, that is, from near-surface layers.
Recently, the detection of near-surface fluorescence with the help of the detection of what is called xe2x80x9cevanescent radiationxe2x80x9d in optical sensors has proven effective. The following physical principle is exploited for this:
When a ray of light strikes an interface between a medium with a higher refractive index and one with a lower refractive index, total internal reflection can occur if the angle of incidence of the ray of light with respect to the surface normal is larger than the critical angle of total internal reflection ax, which is determined by
sin xcex1=Error!xe2x80x83xe2x80x83(1)
(where n2=index of refraction of the medium with a lower one, and n1=index of refraction of the medium with a higher one). Rays of light that strike the interface at an angle larger than xcex1 cannot leave the medium with the higher refractive index, according to considerations of geometrical optics.
And vice-versa, it is not possible according to geometrical optics for a ray of light to penetrate from the medium with a lower index of refraction into the one with a higher index, if it propagates in the medium with the higher refractive index at an angle to the interface that is larger than the critical angle of total internal reflection xcex1.
However, more precise electromagnetic considerations show that the field strength of the light wave striking the interface from the medium with the higher refractive index and being totally internally reflected does not drop abruptly to zero at the interface to the medium with the lower refractive index, but rather decreases exponentially from its value in the medium with the higher refractive index as a function of the distance from the interface. Typical decay constants for this exponential decline in the field strength lie in the range of the wavelength of the incident light. Thus despite total internal reflection, a certain portion of the light enters the medium with the lower refractive index.
Likewise, light from a location in the medium with the lower refractive index that is near the interface can also enter the medium with the higher refractive index at an angle that is greater than the critical angle of total internal reflection xcex1.
This effect is exploited in optical fiber sensors. In these sensors, receptor molecules are immobilized on the surface of the fiber. Exciting light, usually laser light, is launched into the fiber. The laser light propagates along the fiber by total internal reflection. It also passes the regions of the fiber on whose surface the molecules able to fluoresce are bound. Due to the evanescent field of the exciting light at the surface of the fiber, molecules able to fluoresce can be excited. In the bound state, the distance of the molecules from the surface of the fiber is only a few nanometers, so that the exponential drop of the exciting light""s field strength has little effect. Therefore, the molecules bound to the surface are effectively excited by the exciting light. The light emitted by the molecules can in turn be launched into the fiber, in part at angles at which the fluorescence light, once inside the fiber, propagates along the fiber due to total internal reflection. The propagated portion of fluorescence light from the bound molecules can then be detected at one end of the fiber.
A disadvantage of optical fiber sensors that detect evanescent radiation is that the efficiency with which they can collect (capture and transmit) emitted fluorescence photons is very limited. In particular, the sensitivity of such an arrangement is not sufficient to detect individual fluorescence molecules.
The object of the invention is to improve the light-collecting efficiency for light launched evanescently into a medium with a higher refractive index.
According to the present invention, this object is achieved by an optical arrangement.
The optical arrangement according to the invention can detect both light that has been generated on an interface, for example due to chemoluminescence, and also light scattered on the interface, for example fluorescence light due to excitation by a light source.
The light thus generated is collected by a light-collecting device. Besides the optical waveguide according to the invention, this can also include additional lenses, mirrors, filters, and other usual optical components.
The light collected by the light-collecting device is detected by the detection device. This also can include, in addition to an ordinary detector, additional lenses or filters for directing the light onto the detector.
The first end face may be entirely planar, or only flat in a region where the sample is located. The interface in front of which the sample is located can be formed directly by the first end face itself. Or alternatively, the first end face can be optically coupled to an interface between the sample medium and the medium with a high index of refraction. In this case, the medium with the high index of refraction can be the medium of an microscopic slide that is coupled to the first end face with the help of an immersion oil with a high refractive index. In the latter case, the diameter of the microscopic slide and the dimensions of the first end face should be selected so that light emitted by the sample, and radiated into the half-space facing the medium with a high index of refraction, is not prevented from entering the first end face by purely geometrical limitations.
If light is emitted by the sample near the interface, for example by generating fluorescence light, then a portion of the light enters the optical waveguide through the first end face, and propagates at an angle (with respect to the surface normal of the first end face at the site of the sample) within the optical waveguide which is larger than the critical angle of total internal reflection corresponding to the ratio of indices of refraction of the medium with a high index of refraction and the sample medium. In fiber sensors, this xe2x80x9cevanescentxe2x80x9d radiation could only propagate in part to the end of the fiber, and thus onto a detector. According to the invention, the arrangement and construction of the shell surface ensures that the evanescent radiation at the shell surface is totally reflected back into the optical waveguide. This can be done on the one hand by means of total internal reflection, or on the other hand by an appropriate mirror-coating of the shell surface. The evanescent radiation can then be directed onto a detection device and be detected by the latter according to prior art.
In this way, the evanescent portion of the radiation can be detected essentially entirely. Since also classical radiation can be detected, i.e. light that propagates in the optical waveguide at an angle (with respect to the surface normal mentioned above) that is smaller than the critical angle of total internal reflection corresponding to the ratio of indices of refraction of the medium with a high index of refraction and the sample medium, almost the entire radiation of a half-space can be detected.
In an advantageous embodiment, the first end face is flat. The shell surface adjacent to this is at such an angle to the first end face that the light emitted by the sample is totally reflected at the shell surface. In this way, no-loss reflection can be achieved. Further, a special mirror-coating of the shell surface is not needed.
In an advantageous embodiment of the invention, the arrangement and construction of the shell surface are selected so that essentially only the evanescent portion strikes the shell surface and is totally reflected at it. The classical radiation, on the other hand, leaves the optical waveguide unreflected.
This permits an extremely efficient separation of evanescent and classical radiation. Evanescent portions of radiation can only originate from sources close to the interface. Thus the light of a sample emitted near the surface can be detected very selectively, which is essential for optical biosensors.
In an advantageous embodiment of the invention, the optical waveguide is constructed to be symmetrical about an axis, and the sample is located on or in the immediate vicinity of the axis. The shell surface can then be a section of a paraboloid of revolution, with the sample being located near to the focal point. If the light emitted comes from the sample, and thus from the focal point of the paraboloid of revolution, it is collimated by reflection at the shell surface. Light thus collimated can be collected and detected in a simple way. If the second end face is flat, the collimated light will also emerge from the optical waveguide in parallel. It can then be handled especially easily. Furthermore, the radial distance of the ray of light emerging from the second end face depends in a well-defined way on the angle of entrance into the first end face.
In an advantageous embodiment of the invention, the shell surface is a section of an ellipsoid of revolution, with the sample being arranged near a focal point of the ellipsoid of revolution on or in front of the first end face.
This improvement is especially suitable for collecting and detecting classical and evanescent radiation in the second focal point of the ellipsoid of revolution beyond the second end face of the optical waveguide. In this way, a maximum efficiency of light collection can be achieved in an elegant fashion at sensor which is almost a point in size.
The shell surface can also be constructed as the outer surface of a frustum of a cone. This permits simple manufacturing of the optical waveguide. A frustum-shaped shell surface has also proven to provide an improved imaging quality, compared with a shell surface in the shape of a paraboloid of revolution, since deviations of the site of the radiation source from the axis have less effect on the sharpness of the image.
In another embodiment, the second end face is of convex construction, and acts as a focusing lens for the totally-reflected light. Thus the latter can be focused directly onto a point. The detection device can be located at this focal point, for example. This permits setting up the light-collecting device, which consists essentially of the optical waveguide.
In an advantageous embodiment of the invention, a light-absorbing aperture is arranged between the second end face and the detection device in such a way that it absorbs the classical radiation portion of the light emerging from the optical waveguide selectively. In most designs of the shell surface, the evanescent and classical portions of the radiation emerge at different places and/or at different angles from the second end face. An aperture is then able to absorb the classical radiation selectively from a mixture of classical and evanescent radiation, and only allow the evanescent radiation to pass. Selective detection of only the evanescent radiation in turn permits the selective detection of light emitted near the interface, which is important for optical biosensors.
Alternatively, the detection device can be constructed so that it detects classical and evanescent radiation separately. This can be done with the help of a detector with spatial resolution, for example. The separate detection of evanescent and classical radiation permits a further differentiation between light scattered near and far from the surface. The distance of the sample from the first interface can also be determined by this.
For detecting fluorescence light from a sample, the optical arrangement possesses a light source for irradiating the sample. The light can be transmitted through the optical waveguide onto the sample, for example by injecting it at the second end face, or injecting it in such a way that it excites the sample evanescently. As a rule, however, it is coupled into the waveguide at the sample end, and thus enters the optical waveguide through the first end face. In an advantageous improvement, an absorption device is located between the optical waveguide and the detection device, which absorbs the light from the light source so that it does not reach the detector. In this way, a disturbing influence of the light from the light source on the detection is prevented. This is essential for achieving the desired extremely high sensitivities, which permit detection of individual fluorescent molecules.
In an advantageous embodiment of the invention, a thin separating window made of a medium with a higher index of refraction is arranged between the first end face and the interface. Typically, this can be an microscopic slide or a microscopic cover sheet. The thickness of the separating window is substantially less than the minimum dimension of the first end face, so that the main portion of the evanescent radiation can enter the optical waveguide. This is particularly favorable for routine performance of analyses. Typically, receptor molecules are immobilized on a microscopic slide""s surface in such cases. The microscopic slide is coupled optically to the first end face by an immersion oil on the opposite surface. In this way, direct contact between the optical waveguide and the sample, and thus contamination of the first end face, and resultant background signals, are avoided.
In an advantageous embodiment of the invention, the microscopic slide is mounted so that it can shift with respect to the optical waveguide in parallel to the first end face. The optical coupling between the optical waveguide and the microscopic slide remains even if the microscopic slide is shifted, due to the immersion oil. Thus it is possible to search the surface of a microscopic slide to see whether an individual or a few individual molecules sought have bound to the receptor molecules immobilized on it. This is important if extreme sensitivities are to be achieved, since the concentrations of the molecules sought are so small that no molecule sought may be bound to a receptor molecule in a predetermined small observation area.
In an advantageous embodiment of the invention, the microscopic slide is part of a flow cell. This permits automation of measurements and the analyzing of continuous flows of samples.
Advantageous embodiments of the invention are identified in the dependent claims.